Article Figures & Data

Figures

Organotellurium compounds chemosensitize S. cerevisiae AD strains overexpressing the C. albicans efflux pumps CaCdr1p, CaCdr2p, and CaMdr1p to fluconazole (FLC). Columns labeled “fluconazole (−)” show the growth of serial 5-fold dilutions of the indicated S. cerevisiae strains on YPD agar in the absence of FLC; columns labeled “fluconazole (+)” show the growth of the same serial dilutions of the indicated strains on YPD medium in the presence of subinhibitory concentrations of FLC (∼1/2 to ∼1/4 of their respective MICs). Cells grown on YPD with or without FLC only or on 0.5% DMSO were used as positive controls. Rows 1, 2, 3, 4, and 5 show the growth of the same cells in the presence of 100 μM compounds 1 to 5 (34, 35, 36, 38, and 40 μg/ml, respectively).

Effect of organotellurium compounds 1 to 4 on the intracellular accumulation of R6G in S. cerevisiae AD/CaCDR1. Panels A to D and panels I to L show the bright-field images of the corresponding fluorescence microscopy images shown in panels E to H and M to P. AD/CaCDR1 cells preloaded with R6G were incubated under the following conditions: A and E, 0.2% glucose; B and F, no glucose; C and G, 0.2% glucose + DMSO; D and H, 0.2% glucose + compound 1; I and M, 0.2% glucose + compound 2; J and N, 0.2% glucose + compound 3; and K and O, 0.2% glucose + compound 4. Panels L and P show cell images for the R6G-preloaded sensitive control strain S. cerevisiae AD in the presence of 0.2% glucose. The pump-inhibitory effects of compounds 1 to 5 were tested at 100 μM (34, 35, 36, 38, and 40 μg/ml, respectively).

Effect of organotellurium compounds 1 to 5 on the intracellular accumulation of Nile red in S. cerevisiae AD/CaMDR1. The panels A to D and I to L show the bright-field images of the corresponding fluorescence microscopy images shown in panels E to H and M to P. AD/CaMDR1 cells preloaded with Nile red were incubated under the following conditions: A and E, PBS (pH 7.2) without any compound; B and F, + 0.5% DMSO; C and G, + compound 1; D and H, + compound 2; I and M, + compound 3; J and N, + compound 4; and K and O, + compound 5. Panels L and P show cell images for the Nile red-preloaded sensitive control strain S. cerevisiae AD in PBS (pH 7.2) without the addition of any compound. The pump-inhibitory effects of compounds 1 to 5 were tested at 100 μM (34, 35, 36, 38, and 40 μg/ml, respectively).

Quantification by flow cytometry of the CaCdr1p pump-inhibitory effects of organotellurium compounds 1 to 5. The bars indicate the percent R6G accumulation relative to the sensitive control strain AD/pABC3, which was set to 100%, in the absence (■) or presence () of 0.2% glucose of R6G-preloaded AD/CaCDR1 cells incubated in the presence of DMSO, 10 μM FK506 (8.0 μg/ml), or 100 μM compounds 1 to 5 (34, 35, 36, 38, and 40 μg/ml, respectively). The data represent the means ± the standard errors from three independent experiments (*, P < 0.05).

Quantification by flow cytometry of the CaMdr1p pump-inhibitory effects of organotellurium compounds 1 to 5. Gray bars indicate the percent Nile red accumulation relative to the sensitive control strain AD/pABC3 in the presence of PBS (pH 7.2), which was set to 100% of Nile red-preloaded AD/CaMDR1 cells incubated in the presence of buffer only (PBS, pH 7.2), buffer including the vehicle control of 0.5% DMSO, or buffer plus 100 μM concentrations of compounds 1 to 5 (34, 35, 37, 38, and 40 μg/ml, respectively), dissolved in DMSO, as indicated. The data represent the means ± the standard errors from three independent experiments (*, P < 0.05 compared to AD/CaMDR1).

Test of the cytotoxicity of organotellurides. Three different mammalian cell lines were used to test the cytotoxicity (see Materials and Methods for further details) of the organotellurium compounds 1 to 5; J774 (A [macrophages]), HFF (B [fibroblasts]), and HaCaT (C [keratinocytes]). Each compound was tested at three 25, 50, and 100 μM concentrations (i.e., compound 1 was tested at 8, 17, and 34 μg/ml, compound 2 at 9, 18, and 35 μg/ml, compound 3 at 9, 18, and 36 μg/ml, compound 4 at 9, 19, and 38 μg/ml, and compound 5 at 10, 20, and 40 μg/ml, respectively). The data are the means ± the standard errors from three independent experiments.

Organotellurium compounds 1 to 5 chemosensitize C. albicans clinical FLC-resistant isolates. “Fluconazole (−)” columns show the growth of serial 5-fold dilutions of the indicated C. albicans isolates on Sabouraud agar in the absence of FLC; the fluconazole-positive columns show the growth of the same serial dilutions of the indicated isolates on Sabouraud medium in the presence of subinhibitory concentrations of FLC (i.e., ∼1/5 of the MICFLC for C. albicans isolates 95-142 and 12-99 and almost as much FLC as its MICFLC for C. albicans 96-25). Cells grown on Sabouraud agar with or without FLC only or 0.5% DMSO were used as positive controls, and cells grown on 10 μM FK506 (i.e., 8.0 μg/ml) were used as negative controls. Rows 1, 2, 3, 4, and 5 show the growth of the same cells in the presence of 100 μM concentrations of compounds 1 to 5 (i.e., 34, 35, 37, 38, and 40 μg/ml, respectively).

↵a MIC values were determined by a broth microdilution assay according to CLSI protocol M27-A3 (2008) using 50% growth reduction as the cutoff for the reported MIC values. MIC1, the MIC of the compound or FLC alone; MIC2, the MIC of the compound or FLC in the presence of FLC or compound, respectively. FIC, fractional inhibitory concentration; FICI, fractional inhibitory concentration index. *, values were converted from μM to μg/ml.